Construction Of Electrochemical Aptasensing Technique For High-sensitive Detection Of Circulating Tumor Cells

BackgroundLiquid biopsy is the method to detect circulating tumor cells(CTCs),circulating tumor DNA and RNA,exosomes and other tumor-related substances in body fluid.Liquid biopsy has the advantages of minimal trauma,low cost,repeatable sampling,and real-time detection.Due to epithelial-mesenchymal transition or external stimulation(such as diagnosis and treatment),CTCs fall off from tumor in situ or metastasis,invade and colonize to other tissues and organs through blood and lymphatic circulatory system,and proliferate massively,leading to tumor metastasis and recurrence.The detection of CTCs can assist the early diagnosis of breast cancer,lung cancer,colorectal cancer and other cancers.CTCs detection can also help determine the prognosis,monitor the development of tumors and the effects of treatments,and develop individualized treatments.At the same time,CTCs can also be used as a therapeutic target.Because of the rarity of CTCs in the blood and their heterogeneity,it brings great challenges to the detection of CTCs.At present,various techniques including flow cytometry and immunofluorescence have been applied to the CTCs detection.However,these methods are complicated,time-consuming and rely on precision instruments in different degree.Therefore,it is urgent to establish a high-sensitive and specific,rapid,simple and low-cost detection method.Biosensors have become a research hotspot in the field of medicine because of their small size,low cost,simple operation and high sensitivity.Based on the electrochemical biosenors,researchers have achieved trace detection of biological samples such as proteins,cells,bacteria,viruses,and small molecules.Aptamer is DNA or RNA containing 20-100bases,which specifically recognizes and binds to the targets by spatial structures such as hairpin,pseudoknot,and G-quadruplex.Aptamer is an ideal biological probe that can bind to the target besides antibody,and is vividly referred to as"chemical antibody".Aptamer has the advantages of high affinity,easy synthesis and modification,good stability,no toxicity,and low or no immunogenicity,so it is suitable for the identification and capture of target cells.Isothermal amplification techniques such as rolling circle amplification(RCA)can extend the nucleic acid chain exponentially at constant temperature.It has the characteristics of convenient design and high amplification efficiency.Therefore,RCA can be used for signal amplification to improve the sensitivity of biosensors.In this study,K562 cells were used as a target.One aptamer was designed as capture probe while the other one was designed as signal probe.RCA was combined with signal probe to amplify the electrochemical signal and they were labeled on the target cells.A novel electrochemical biosensor for the detection of CTCs was constructed based on dual-aptamer sandwich recognition.On the other hand,in order to improve the detection performance,more and more nanomaterials are applied to the biosensors.Among them,metal nanoparticles such as Au nanoparticles(Au NPs)have the characteristics of good biocompatibility,high conductivity,large active area and catalytic activity.Therefore,in order to develop a more convenient and efficient method of CTCs detection,Au NPs were modified on the biosensing platform to fix more aptamers by increasing the active area of the reaction interface.Due to the specific recognition and binding between aptamers and target cells,which lead to the conformational change of the double-stranded hybrids and the output of electrochemical signal,a highly sensitive and label-free method for the detection of CTCs was fabricated.Objectives1.To construct a novel electrochemical biosensor based on the strategy of dual-aptamer sandwich recognition and RCA to amplify electrochemical signal.The proposed biosensor can realize the high sensitivity and specificity detection of CTCs,and help for the early diagnosis and treatment of tumors.2.To construct a label-free and"signal-off"electrochemical aptasensor for high-specific and sensitive CTCs detection based on the high affinity between aptamers and target cells and Au NPs to enlarge the active area of the biosensing platform.The developed aptasensor provides a non-invasive method for the early diagnosis and prognosis of tumors.Methods1.Construction of electrochemical biosensing technique based on dual-aptamer sandwich recognition and RCA and its application in the detection of CTCs.(1)The fluorescence-labeled aptamers and random sequence were incubated with K562cells respectively,and then the cells were analyzed by flow cytometry.The specificity of the binding capability between aptamers and K562 cells was verified through the changes in fluorescent intensity.(2)One aptamer which labeled by fluorescent dye,was incubated with K562 cells and excessive amounts of the other label-free aptamer.The different binding sites between the two aptamers and K562 cells were investigated through the changes in fluorescent intensity.It provided a basis for the next step to construct a biosening platform based on the dual-aptamer sandwich recognition.(3)The effect of RCA was verified by polyacrylamide gel electrophoresis.(4)The modification of the electrode was verified by cyclic voltammetry(CV)and electrochemical impedance spectroscopy(EIS).(5)To obtain the best detection performance,the important reaction conditions were optimized such as the concentration of the capture probe assembled on the biosensor,the time and temperature for the capture probe to react with K562 cells,and the time and temperature for the signal probe to react with K562 cells.(6)The detection performance of the proposed biosensor was determined under the optimal conditions.The linear equation,linear range and limit of detection(LOD)were obtained.(7)Different types of tumor cells were determined under the optimal conditions to verify the specificity of the proposed biosensor.To verify the reproducibility of the proposed biosensor,the same batch of modified electrodes was used to quantify K562 cells.To verify the stability of the proposed biosensor,a batch of assembled electrodes was stored.A group of assembled electrodes were carried out to quantify K562 cells every 7 days.(8)To preliminarily verify the potential clinical applications of the proposed biosensor,the recovery test was carried out using the serum of healthy people to mimic clinical samples.(9)To preliminarily evaluate the feasibility of K562 cells for follow-up research,K562cells captured on the biosensor were recovered and cultured continuously using specific enzyme to degrade aptamers.The growth of K562 cells was observed by microscopy in one week.2.Construction of CTCs detection technique based on label-free and"signal-off"electrochemical aptasensing(1)Fluorescence-labeled aptamers and random sequence were incubated with K562 cells respectively,and then the cells were detected through flow cytometry.The changes of fluorescent intensity were analyzed to verify the specific binding capability between aptamers and K562 cells.(2)To validate whether Au NPs and double-stranded hybrids were successfully modified on the electrode,the two-dimensional,three-dimensional morphology and element content of the modified electrode were analyzed by FE-SEM,AFM and EDS,respectively.(3)The effect of Au NPs to increase the active area of the biosensor was verified by calculating the active areas of bare gold electrode and Au NPs-modified electrode.(4)The fabrication of the label-free aptasensor was verified by CV and EIS.(5)The concentration of methylene blue-modified aptamers,the reaction time and temperature between biosensor and K562 cells were optimized to obtain the optimal performance of biosensor.(6)The linear equation,linear range and LOD of the fabricated biosensor were obtainedunder the optimal conditions.(7)Under the optimal conditions,the control tumor cells and interferences such as PBS,FBS and BSA were tested respectively to verify the specificity of the biosensor.The same batch of electrodes was used to determine K562 cells to verify the reproducibility of the biosensor.To evaluate the stability of the biosensor,a batch of assembled electrodes was stored,while a group of electrodes were used to determine K562 cells every 6 days.(8)The potential clinical applications of the biosensor was preliminarily validated through recovery test using serum from healthy human to mimic clinical samples.Results1.An electrochemical biosensor based on dual-aptamer sandwich recognition and RCA was successfully constructed.The optimal conditions of the electrochemical biosensor for K562 cells detection were investigated.The linear range of the biosensor was 1×10~2-1×10~5 cells/m L,and the LOD was 25 cells/m L.At the same time,the electrochemical biosensor had good specificity,reproducibility and stability,and less damage to K562 cells.In addition,the recovery test showed that the biosensor had the potentials of clinical applications.2.A label-free and"signal-off"electrochemical aptasensor was successfully fabricated.The experimental conditions of the proposed label-free electrochemical aptasensor for K562cells detection were optimized.The linear range of the aptasensor was 1×10~2-1×10~6cells/m L,while the LOD was 23 cells/m L.The fabricated aptasensor had good specificity,reproducibility and stability.In addition,the recovery test of the aptasensor is explored by mimicking clinical samples,which showed that it had the potentials of clinical applications.Conclusions1.An electrochemical biosensor was successfully constructed to realize the highly sensitive detection of CTCs based on dual-aptamer sandwich recognition and RCA.This method provides a promising method for the construction of universal biosensors based on different CTCs,and helps for the early diagnosis and prognosis of tumor.2.A label-free and"signal-off"electrochemical aptasensor was constructed to achieve high-sensitive detection of CTCs based on combination with nanotechnology and clinical laboratory diagnostic methods.The linear range of label-free aptasensor is wider,the detection process is simpler and the detection time is shorter.The proposed electrochemical aptasensor provides a non-invasive detection method for early diagnosis of tumor.